Subcellular and single-molecule imaging of plant fluorescent proteins using total internal reflection fluorescence microscopy (TIRFM).

Total internal reflection fluorescence microscopy (TIRFM) has been proven to be an extremely powerful technique in animal cell research for generating high contrast images and dynamic protein conformation information. However, there has long been a perception that TIRFM is not feasible in plant cells because the cell wall would restrict the penetration of the evanescent field and lead to scattering of illumination. By comparative analysis of epifluorescence and TIRF in root cells, it is demonstrated that TIRFM can generate high contrast images, superior to other approaches, from intact plant cells. It is also shown that TIRF imaging is possible not only at the plasma membrane level, but also in organelles, for example the nucleus, due to the presence of the central vacuole. Importantly, it is demonstrated for the first time that this is TIRF excitation, and not TIRF-like excitation described as variable-angle epifluorescence microscopy (VAEM), and it is shown how to distinguish the two techniques in practical microscopy. These TIRF images show the highest signal-to-background ratio, and it is demonstrated that they can be used for single-molecule microscopy. Rare protein events, which would otherwise be masked by the average molecular behaviour, can therefore be detected, including the conformations and oligomerization states of interacting proteins and signalling networks in vivo. The demonstration of the application of TIRFM and single-molecule analysis to plant cells therefore opens up a new range of possibilities for plant cell imaging.